4. Comparison of sP(EO-stat-PO) with Isocyanate and Acrylate end groups as respective cross ...

148 Pages·2011·3.43 MB·English

by  Anandhan Dhanasingh

sP (EO-stat-PO) - Glycosaminoglycans
(GAGs) Hybrid-Hydrogels for Medical
Applications

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der
RWTH Aachen University zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Anandhan Dhanasingh, B.E., M.Sc.,

aus Chennai, Indien

Berichter: Univ.Prof. Dr. Martin Möller
Prof. Dr. Jürgen Groll

Tag der mündlichen Prüfung: 01.12.2011

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
-ஔவையார்

What has learnt is a handful, and what has still to acquire is wide as the world

- Auvaiyar
(Before 9th century, India)
Table of Contents
Table of Contents
Acknowledgement...................................................................................................... ii
Summary..................................................................................................................... iv
List of abbreviations................................................................................................... vii

1. Introduction
1.1 Hydrogels as DDS and TE Scaffolds................................................................. 1
1.2 Description of this thesis.................................................................................... 3
1.3 References.......................................................................................................... 4

2. Literature Overview
2.1 Hydrogel........................................................................................................... 5
2.1.1 Introduction.................................................................................................. 5
2.1.2 Classification of hydrogels........................................................................... 5
2.1.3 Properties of hydrogels................................................................................. 8
2.1.4 Classification of water in hydrogels............................................................. 11
2.1.5 Common applications of hydrogels.............................................................. 12
2.1.6 Summary of the important physico-chemical parameters and properties
of hydrogels in general................................................................................. 13

2.2 Hydrogels as biomedical materials
2.2.1 Introduction................................................................................................ 14
2.2.2 Hydrogels in drug delivery system............................................................. 15
2.2.3 Hydrogels as tissue engineering scaffolds.................................................. 19

2.3 Extra-cellular matrix (ECM)
2.3.1 Introduction................................................................................................ 22
2.3.2 Classes of ECM.......................................................................................... 23
2.3.3 Component of CM...................................................................................... 23

2.4 GAGs and their importance in connective tissue/ECM
2.4.1 Introduction................................................................................................ 24
2.4.2 Classification of GAGs............................................................................... 24
2.4.3 Properties of GAGs.................................................................................... 25
2.4.4 Proteoglycans.............................................................................................. 26
2.4.5 Hyaluronan................................................................................................. 27

2.5 References......................................................................................................... 38

3 NCO-sP(EO-stat-PO) as cross-linker to HA
3.1 Introduction...................................................................................................... 4 3
3.2 Materials........................................................................................................... 44
3.3 Hydrogels preparation...................................................................................... 44
3.4 Contact angle measurements............................................................................ 45
3.5 Equilibrium water content measurements........................................................ 45
3.6 Differential scanning calorimetry..................................................................... 46
3.7 FT-IR spectroscopy.......................................................................................... 46
3.8 Cryo-FESEM.................................................................................................... 46
3.9 Rheological analysis......................................................................................... 46
3.10 In-vitro stability test in PBS............................................................................. 47
Table of Contents
3.11 Enzymatic degradation.................................................................................... 47
3.12 Statistical nalysis.............................................................................................. 47
3.13 Results and discussions.................................................................................... 48
3.14 Conclusion........................................................................................................ 58
3.15 References........................................................................................................ 60

4 Comparison of sP(EO-stat-PO) with isocyanate and acrylate end groups
as respective cross-linker for unmodified and thiol modified
Hyaluronic acid
4.1 Introduction...................................................................................................... 62
4.2 Materials........................................................................................................... 63
4.3 Hydrogel preparation........................................................................................ 63
4.4 Contact angle measurements............................................................................ 64
4.5 Equilibrium water content measurements........................................................ 64
4.6 Differential scanning calorimetry..................................................................... 65
4.7 FT-IR spectroscopy.......................................................................................... 65
4.8 RAMAN spectroscopy..................................................................................... 65
4.9 Cryo-FESEM.................................................................................................... 65
4.10 Rheological analysis......................................................................................... 65
4.11 In-vitro stability in PBS.................................................................................... 66
4.12 Enzymatic degradation..................................................................................... 66
4.13 Statistical nalysis.............................................................................................. 66
4.14 Results and discussion...................................................................................... 67
4.15 Conclusion........................................................................................................ 77
4.16 References........................................................................................................ 78

5 NCO-sP(EO-stat-PO) as universal cross-linker to GAGs
5.1 Introduction...................................................................................................... 81
5.2 Materials........................................................................................................... 82
5.3 Hydrogel preparation........................................................................................ 82
5.4 Contact angle measurements............................................................................ 83
5.5 Equilibrium water content measurements......................................................... 83
5.6 FT-IR spectroscopy........................................................................................... 83
5.7 Cryo-FESEM.................................................................................................... 83
5.8 Rheological analysis......................................................................................... 84
5.9 In-vitro stability test in PBS............................................................................. 84
5.10 Results and discussions.................................................................................... 84
5.11 Conclusion........................................................................................................ 90
5.12 References........................................................................................................ 91

6 PEG based hydrogels in drug delivery system
6.1 Introduction...................................................................................................... 92
6.2 Materials........................................................................................................... 93
6.3 Hydrogel preparation together with drug encapsulation.................................. 93
6.4 Drug release by diffusion controlled mechanism............................................. 95
6.5 Equilibrium water content measurement of hydrogels without the model
drug................................................................................................................... 95
6.6 In-vitro drug release system............................................................................. 95
6.7 Results and discussions..................................................................................... 96
6.8 Conclusion........................................................................................................ 101
6.9 References......................................................................................................... 102
Table of Contents
7 Poly-saccharide based covalently linked multi-membrane hydrogels
7.1 Introduction..................................................................................................... 103
7.2 Materials.......................................................................................................... 104
7.3 Single layered bulk hydrogel preparation........................................................ 104
7.4 Preparation of multi-membrane hydrogels….................................................. 105
7.5 Drug loading procedure……………............................................................... 105
7.6 Rheological analysis of the hydrogels...…...................................................... 106
7.7 Drug release study………………………………………............................... 106
7.8 Results and discussions……………………………....................................... 106
7.9 Conclusion and outlook…............................................................................... 113
7.10 References....................................................................................................... 114

8 Hydrogels as Cell-Scaffold
8.1 Introduction..................................................................................................... 115
8.2 Materials.......................................................................................................... 116
8.3 Cell culture....................................................................................................... 117
8.4 Hydrogel preparation-Cell encapsulation........................................................ 117
8.5 Cell culture on PEG electrospun fibres-encapsulated in hydrogel.................. 117
8.6 Viability staining.............................................................................................. 118
8.7 Immunological staining................................................................................... 118
8.8 Optical and fluorescence microscopy.............................................................. 118
8.9 Results and discussions.................................................................................... 118
8.10 Conclusion....................................................................................................... 120
8.11 References....................................................................................................... 122

Appendix
A1 Preparation of hydrogels (free form hydrogels).............................................. x
A1a Hydrogels in constricted environment............................................................. xi
A2 Swelling experiments of hydrogels................................................................. xi
A3 Sample preparation for FT-IR and RAMAN spectroscopy............................. xii
A4 Contact angle measurement by goniometer..................................................... xii
A5 Differential scanning calorimetry.................................................................... xii
A6 Cryo-FESEM................................................................................................... xiii
A7 Rheological analysis........................................................................................ xiii
A8 Stability test in PBS......................................................................................... xiv
A9 Enzymatic degradation study........................................................................... xiv
A10 Quantification of drug deliverd by UV-spectrophotometer............................. xiv
A11 Flory-Rehner calculation................................................................................. xv
A12 Diffusion co-efficient calculation.................................................................... xvii

Curriculum Vitae xix
Acknowledgement

Acknowledgement

The success of a Ph.D thesis depends not only on the performance of the student but
also on the relationship between the student and his supervisor. In the worst case it is a
nightmare that both the student and the supervisor want to get over with. In my case, I was
very fortunate to have Prof. Dr. Jürgen Groll as my supervisor who became my friend and
moral advisor in the last three years. With my knowledge in the field of mechanical and
biomedical engineering, it was really difficult for me to get the chemistry behind my Ph.D
thesis and it was Prof. Dr. Jürgen Groll who took much pain and never fed up in teaching me
the basics of chemistry. It is indeed not enough to simply thank him for giving me such an
interesting topic, for all of his help and financial support.

I thank Prof. Dr. Martin Möller for giving me the permission to use all the facilities
of DWI e.V and Institute of technical and macromolecular chemistry of Aachen University of
Technology (RWTH) in performing all the practical part of my thesis work from May 2006 to
August 2009 and for extending the financial support in the form of stipend that helped me to
do part-time job at the weekends.

My special thanks to Prof. Dr.Doris Klee who welcomed me in her research group to
make my master thesis that ultimately showed me the way to my PhD. Thanks to Dr. Jochen
Salber, project leader in Prof. Dr. Doris Klee’s group, for their supervision in a project that
was done in co-operation with Uni-klinik Aachen and for their worthy ideas and timely
encouragement.

I am grateful to Dipl. Chem. Haika Hildebrandt and Mrs. Michaela Meuthrath for the
synthesis of Isocyanate starPEG. FTIR and RAMAN spectroscopy would not have been
possible without the help of Dr. Walter Tillmann and I thank him. My thanks further goes to
Mr. Stefan Rütten and Mrs. Claudia Formen for their assistance in FESEM and UV-
spectrophotometry respectively.

My sincere gratitude to Dipl.Biotech. Vera Schulte for the cell experiments on my
hydrogels. I would like to thank Dr. Arthur Henke for performing some of the dynamic light
i
Acknowledgement
scattering experiments, although those results were not included in this thesis and also for his
discussions in the statistical analysis of my results.

I thank Prof. Dr. Crisan Popescu and Dipl. Ing. Daniel Istrate for their support in
performing the thermal analysis of my hydrogels. I further thank Dipl. Ing. Dora Alves for her
introduction to rheometer.
I would like to extend my thanks to all the members of Prof. Dr .Juergen Groll’s group
especially to Mrs. Konstantina Dyankova and Mrs. Smriti Singh for their moral support.

Finally I would like to thank my parents for initiating me to do Ph.D and my wife for
her deep cooperation when I had to spend long hours in the lab and during the weekends of
my part-time job and I dedicate this work to them.

A timely benefit, -though thing of little worth,
the gift itself, -in excellence transcends the earth.

-- Thiruvalluvar
(Before 9th century, India)

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Summary
Summary

The primary focus of this work is to develop a highly stable hydrogel system as a
depot for drug delivery and as material for tissue engineering scaffolds. Two separate cross-
linkers namely six arm star shaped (EO-stat-PO) prepolymers with isocyanate endgroups
[NCO-sP(EO-stat-PO)] and six arm star shaped (EO-stat-PO) prepolymers with acrylate
endgroups [Acr-sP(EO-stat-PO)] were prepared to gel unmodified and thiol-modified bio-
polysaccharides respectively. In particular we discuss three types of hydrogel systems for the
above mentioned bio-applications namely, hydrogels formed by gelation of NCO-sP(EO-stat-
PO) only, i.e., the hydrogel system-1, hydrogel formed by addition of hyaluronic acid to
NCO-sP(EO-stat-PO), i.e. the hydrogel system-2 and hydrogel formed by addition of thiol-
modified hyaluronic acid to Acr-sP(EO-stat-PO), i.e. the hydrogel system-3.

Different than hydrophobic particles or layers, hydrogels are open to water. Water
molecules can diffuse in and out and thus extract a drug, that has been loaded to the hydrogel
rather unrestricted. In systemic application, the organism does not easily recognize hydrogel
particles as foreign and circulation times are rather long. Thus hydrogel particles can
overcome delivery barriers such as early uptake in the liver. Hydrophobic drugs can be loaded
to hydrogels within hydrophobic pockets, while hydrophilic drugs usually have to be bound
covalently and released by bond splitting to prevent premature drug release. In order to ensure
that the hydrogels cannot be harmful, degradability is essential. In the systems observed here,
degradation will yield fragments not larger than the prepolymers, whose molecular weight
was chosen to allow excretion via the renal system. For the application as scaffold for cell
culture and tissue engineering, the hydrogels have the advantage that they interact little with
the cells but can be equipped with biological components such as peptides to mimic the
natural extracellular matrix (ECM). Biocompatibility, drug-release, but also mechanical
strength of hydrogels are controlled by their degree of swelling, that depends on the chemical
nature and the degree of cross-linking.

Water content and the size of the pores increases with a decrease in cross-linking
density and it is the other way around with the increase in the cross-linking density. As the
network is formed from the hexafunctional sP(EO-stat-PO) as a cross-linker and hyaluronic
acid as a linear polysaccharide, the degree of cross-linking depends on the relative
concentration of the two components. We also studied the effect of different molecular weight
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Summary
of the unmodified hyaluronic acid. Higher molecular weight (longer chains) HA yielded gels
with a higher water content, bigger pore size and lower mechanical strength than the lower
molecular weight (shorter chains). Within the time of the experiment, i.e. up to several weeks,
the hydrogel system-1 showed no degradation. The equilibrium degree of swelling and
internal micro-porous structures are inversely proportional to the concentration of NCO-
sP(EO-stat-PO) of the gelling solution. The hydrogel system-2 showed slow degradation and
the equilibrium degree of swelling and internal micro-porous structures could be varied by the
relative concentrations of the two components. The hydrogel system-3, which is also semi-
synthetic, showed complete degradation. Here cross-links are formed by Michael-type
addition of thiols to acrylates, but degradation occurs via hydrolysis of the acrylate ester
groups.

Testing these hydrogels as drug carrying and delivering vehicles showed for a
hydrophilic drug, i.e. Dexamethasone 21 phosphate disodium salt, a quick and complete
release by a diffusion controlled mechanism. Actually 90% of the drug is released within 8
hours (swelling controlled burst release) followed by a slow release of the rest of the drug by
a diffusion controlled mechanism. It was shown that the release can be retarded by
encapsulating the hydrogel by a silicone tube with one end closed and the other end open for
the controlled release of the drug.

In order to use these hydrogels as cell scaffolds, the primary requirement apart from
high water content and high porous structures is the incorporation of cell attachment sites.
This can be achieved by linking RGD peptides to our hydrogel system. It is a particular
advantage of using NCO-sP(EO-stat-PO) as cross-linker system that the isocyanates can react
not only with the hydroxyl groups of the polysaccharides but also with the amine functional
groups of the peptide sequence. Thus co-addition of the peptides enables biofunctionalisation
in the cross-linking step. Cyto-compatibility tests showed that the bio-functionalised and non-
bio-functionalised cross-linked hydrogels were non-toxic provided complete conversion of the
NCO-groups. The cell experiments using mouse fibroblast L929 showed clearly the cells
adhering on to the RGD decorated sites of the hydrogel coated surfaces. When we added the
cells to the hydrogel precursor prior to complete conversion the NCO-sP(EO-stat-PO) based
hydrogel systems were toxic to cells. In contrast, the Acrylate sP(EO-stat-PO) based hydrogel
system were non-toxic to cells irrespective of the full conversion of the acrylate groups.
Hence, the hydrogel system-1 and 2 cannot be used as cell encapsulation matrix but the cross-
iv